The Ecology of Herbivore-induced Silicon Defences in Grasses

S.E. Hartley*1 and J.L. DeGabriel2

1York Environmental Sustainability Institute, Department of Biology, University of York,

Heslington, York, YO10 5DD, UK.

2Hawkesbury Institute for the Environment, Western Sydney University,

Locked Bag 1797, Penrith, NSW 2751, Australia.

*Corresponding author: sue.hartley@york.ac.uk

Summary

1. Silicon as a defence against herbivory in grasses has gained increasing recognition and has

now been studied in a wide range of species, at scales from individual plants in pots to plant

communities in the field. The impacts of these defences have been assessed on herbivores

ranging from insects to rodents to ungulates. Here we review current knowledge of silicon

mediation of plant-herbivore interactions in an ecological context.

2. The production of silicon defences by grasses is affected by both abiotic and biotic factors

and by their interactions. Climate, soil type and water availability all influence levels of

silicon uptake, as does plant phenology and previous herbivory. The type of defoliation

matters and artificial clipping does not appear to have the same impact on silicon defence

induction as herbivory which includes the presence of saliva. Induction of silicon defences

1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

has been demonstrated to require a threshold level of damage, both in the lab and the field. In

recent studies of vole-plant interactions, the patterns of induction were found to be

quantitatively similar in glasshouse compared with field experiments, in terms of both the

threshold required for induction and timing of the induction response.

3. The impacts of silicon defences differ between different classes of herbivore, possibly

reflecting differences in body size, feeding behaviour and digestive physiology. General

patterns are hard to discern however, and a greater number of studies on wild mammalian

herbivores are required to elucidate these, particularly with an inclusion of major groups, for

which there are currently no data, one such example being marsupials.

4. We highlight new research areas to address what still remains unclear about the role of

silicon as a plant defence, particularly in relation to plant-herbivore interactions in the field,

where the effects of grazing on defence induction are harder to measure. We discuss the

obstacles inherent in scaling up laboratory work to landscape-scale studies, the most

ecologically relevant but most difficult to carry out, which is the next challenge in silicon

ecology.

Key-words: defence induction, insect, physical defences, silica, plant-herbivore

interactions, herbivory, landscape-scale, mammal.

Introduction

Silicon is the second most abundant element in the Earth’s crust and, in grasses at least, may

be present in greater amounts than macro-nutrients, comprising up to 10% dry weight in

2

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

some species (Epstein 1999). Several hypotheses for an ecological role for this extensive

accumulation have been put forward over recent years (Raven 1983; Ma 2004; Massey,

Ennos & Hartley 2007a; Cooke & Leishman 2011), with one of the earliest suggestions being

that silicon was a defence against herbivory. In agricultural systems, it has long been known

that silicon enhances the resistance of crop plants to insect pests (e.g. McColloch & Salmon

1923; Ponnaiya 1951; Sasamoto 1953; Keeping, Meyer & Sewpersad 2013) and that

application of soluble silicon leads to decreased damage by insect herbivores (Goussain,

Prado & Moraes 2005). The effects of silicon augmentation on crop-pest interactions has

been the subject of previous reviews (Keeping & Reynolds 2009; Reynolds, Keeping &

Meyer 2009); here we focus specifically on ecological systems and on the biotic and abiotic

factors which affect the natural induction of silicon-based defences.

In one of the first studies in natural ecosystems, McNaughton and Tarrants (1983) proposed

grass leaf silicification as an “inducible defence” against vertebrate herbivores following their

findings that grasses from grazed areas in African savannas had higher silicon contents than

those from ungrazed ones, and that clipped plants accumulated more silicon than undamaged

ones. However, some grasses had intrinsically higher silicon contents, even when ungrazed,

so the authors concluded silicon was “best viewed as a qualitatively constitutive trait that is,

nevertheless, quantitatively inducible by grazing” (McNaughton & Tarrants 1983). This

work, supported by other early ecological studies (e.g. McNaughton et al. 1985; Brizuela,

Detling & Cid 1986; Cid et al. 1990) suggested that silicon provided wild grasses with an

effective defence against herbivores that could be rapidly mobilised in response to attack

(Karban & Baldwin 1997), contrasting with previous notions that grasses were relatively

undefended (Vicari & Bazely 1993).

3

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

Silicon defences are usually deployed as phytoliths or other forms of amorphous silica (SiO2)

in the leaf epidermis, or deposited in spines, trichomes or hairs on the leaf surface (Currie &

Perry 2007; Hartley et al. 2015; Strömberg, Di Stilio & Song 2016). These structures render

leaves tough and abrasive and therefore physically deter herbivores from feeding (Massey &

Hartley 2006; Massey & Hartley 2009). In addition, they have been shown to reduce the

digestibility of grasses (Shewmaker et al. 1989), act as a structural inhibitor of microbial

digestion in ruminants (Harbers & Thouvenelle 1980; Harbers, Raiten & Paulsen 1981) and

stimulate other plant defence mechanisms (Goussain, Prado & Moraes 2005; Fauteux et al.

2006; Ye et al. 2013). Adverse effects of silicon on rates of herbivory and animal

performance have now been demonstrated on a range of insect herbivores (Massey, Ennos &

Hartley 2006; Massey & Hartley 2009; Reynolds, Keeping & Meyer 2009; Keeping, Miles &

Sewpersad 2014), rodents and lagomorphs (Gali-Muhtasib, Smith & Higgins 1992; Massey

& Hartley 2006; Cotterill et al. 2007; Huitu et al. 2014; Wieczorek et al. 2015a; Wieczorek et

al. 2015b) and ruminants (Massey et al. 2009). Studies on wild mammalian herbivores

remain relatively lacking however, in marked contrast to the numbers of studies on the effects

of silicon on agricultural insect pests (Massey, Ennos & Hartley 2006; Kvedaras et al. 2009;

Reynolds, Keeping & Meyer 2009; Keeping, Miles & Sewpersad 2014).

More recent work has expanded our understanding of silicon induction, i.e. the increase in

silicon accumulation that occurs in plants when they are damaged, and its similarities and

contrasts with other inducible defences. In common with many types of inducible plant

defences, induction of silicon is often greater in response to attack by herbivores than to

artificial clipping (e.g. Massey, Ennos & Hartley 2007b; Quigley & Anderson 2014),

although in contrast to other types of defence (Tanentzap, Vicari & Bazely 2014), the role of

herbivore saliva in the expression of silicon is unclear. It also appears to be non-linearly

4

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

related to both the frequency and intensity of damage, requiring multiple damage events and

a threshold amount of biomass to be removed (Massey, Ennos & Hartley 2007b; Reynolds et

al. 2012). It appears that the response of plant silicon levels to damage, particularly in the

case of clipping, varies with plant species, genotype and phenological stage, as well as

damage intensity (Kindomihou, Sinsin & Meerts 2006; Soininen et al. 2013). Unlike many

induced defences (but see Haukioja & Neuvonen 1985), silicon induction persists for several

months (Reynolds et al. 2012), reflecting the recalcitrant nature of silicon phytoliths, which

are not remobilised once formed (Piperno 2006; Strömberg, Di Stilio & Song 2016), and

hence tend to accumulate as leaf tissue ages. This persistence has consequences for the

impact of induced silicon defences on herbivores, particularly for small mammals where

delayed density-dependent effects drive population dynamics (Lindroth & Batzli 1986;

Ergon, Lambin & Stenseth 2001; Smith et al. 2006; Ergon et al. 2011). A time lag in defence

induction, due to the requirement for persistent herbivory and the long “decay time” of

induced silicon levels, could provide a mechanism for such delayed plant-herbivore

feedbacks (Massey et al. 2008). Despite many experimental demonstrations of the

importance of silicon in plant-herbivore interactions, there are cases where no changes in

plant silicon levels in response to herbivory are observed, as well as examples of herbivores

unaffected by silicon-based induced defences (e.g. Banuelos & Obeso 2000; Redmond &

Potter 2006; Damuth & Janis 2011).

Studies on silicon mediated plant-herbivore interactions now encompass a wide range of

natural grass species and include scales from individual plants in greenhouses to plant

communities in the field (Massey, Ennos & Hartley 2007b; Reynolds, Keeping & Meyer

2009; Soininen et al. 2013), allowing us to ask whether consistent patterns are emerging in its

5

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

accumulation and impact, as well as assess which aspects of silicon induction remain poorly

understood. We aim to address the following questions in this review:

(i) How do biotic (specifically herbivory) and abiotic factors influence the

production of silicon defences by natural grasses?

(ii) How does silicon uptake by these grasses impact on different classes of

vertebrates and invertebrate herbivores?

(iii) Do silicon defences provide a viable hypothesis for explaining population

regulation of wild grazing herbivores?

We review our current state of knowledge around these specific questions and summarise

gaps in our understanding of each of these questions. We also suggest possible approaches

for scaling up laboratory work to landscape-scale studies, an exciting future challenge in the

study of silicon-based defences that is essential for answering the third of these questions. We

focus on grasses as this plant family has been the most comprehensively studied in terms of

ecological aspects of silicon-mediated interactions between plants and their herbivores,

although there is evidence of silicon induction in other angiosperm groups (Hodson et al.

2005; Cooke & Leishman 2011; Katz 2015).

Impact of herbivory: silicon induction varies with the type, amount and

timing of damage

One of the features of silicon-based defences which has been frequently demonstrated is that

herbivory induces silicon accumulation to a greater extent than does artificial clipping (e.g.

Massey, Ennos & Hartley 2007b; Quigley & Anderson 2014). This is particularly the case in

studies of mammalian herbivores, with relatively few studies demonstrating this differential

effect in the case of insect herbivory (but see Gomes et al. 2005; Massey, Ennos & Hartley

2007b). For example, in North American studies, grasses from areas that had been heavily

6

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

grazed by prairie dogs showed elevated concentrations of silicon compared to more lightly

grazed ones, but mechanical defoliation did not induce this response, with silicon levels in

clipped leaves lower than those in unclipped ones (Brizuela, Detling & Cid 1986; Cid et al.

1989; Cid et al. 1990), whereas in other cases, clipping led to induction in some grass

species, but not in others (e.g. McNaughton et al. 1985; Kindomihou, Sinsin & Meerts 2006;

Quigley & Anderson 2014). A recent literature review demonstrated that silicon induction

was highly variable between species and dependent on the frequency and intensity of damage

(see below), but on average, induction was more than twice as great in response to herbivory

than to manual defoliation across 34 species/study combinations (Quigley & Anderson 2014).

Natural herbivory elicits a greater induction of defences than mechanical wounding, (e.g.

Hartley & Lawton 1987; Hartley & Lawton 1991; Valkama et al. 2005; Farmer 2014)

mediated through herbivore-specific molecular and physiological plant responses (e.g. Korth

& Dixon 1997; Reymond et al. 2000). Oral secretions provide herbivore-specific cues for

defence induction in many insects (Hartley & Lawton 1991; Alborn et al. 1997; Bonaventure,

VanDoorn & Baldwin 2011; Tian et al. 2012). Components of insect saliva, plant cell wall

fragments and other cues create a signalling cascade which triggers a defence response,

including the production of the so-called “wound hormones” (jasmonic acid (JA) and

salicylic acid), changes in gene expression and increases in secondary metabolites (Heil &

Ton 2008; Bonaventure, VanDoorn & Baldwin 2011; Stam et al. 2014). Equivalent research

on induced defence responses to vertebrate herbivory is relatively lacking (Walters 2010),

although, Tanentzap et al. (2014) recently provided a breakthrough by demonstrating that

moose and reindeer saliva could counter alkaloid defences produced as a result of a grass-

endophyte mutualism. In the case of silicon defences, there has not yet been any test of

7

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

whether the application of herbivore saliva induces uptake to the same extent as actual

herbivory.

Nevertheless, it is apparent that silicon addition can lead to increased expression of a large

spectrum of inducible defence responses and amplifies the JA-mediated induced defence

response by serving as a priming agent for the JA pathway, whilst JA promotes Si

accumulation (Fauteux et al. 2006; Ye et al. 2013). A better understanding of the

mechanisms underlying silicon induction, the impacts of silicon uptake on other defence

pathways in plants, and the reasons for any observed differences in induction in response to

clipping, insect and vertebrate herbivory would enable us to answer important questions

about the ecological role of silicon. For example, we may gain insights into whether silicon

defences can explain the higher levels of dietary specialisation among insect herbivores and

tight pairwise coevolution between insects and their host plants, which is generally less

common amongst mammals, particularly grazers.

There are other differences between clipping and herbivory relating to the various ways

herbivores feed. Lepidoptera usually feed by shearing off plant material with their incisors,

gramnivorous orthopterans rely on the molar regions of their mandibles to mechanically

disrupt the cell wall, whilst phloem-feeding insects such as aphids use a piercing and sucking

mechanism (Bonaventure 2012). Each of these actions is likely to damage plant cells in a

different way and to a greater extent than would mechanical snipping, which results in a

cleaner cut and less disruption to the plant cells, hence we might expect differences in the

effects of herbivory between different guilds of insects and mammalian herbivores.

8

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

In fact, we still have surprisingly little data on the relative magnitude of silicon induction by

different types of herbivore (but see Quigley & Anderson 2014). It is possible that herbivory

by some species of mammalian herbivores might not result in the induction of chemical or

physical defences, since the speed, pattern and amount of leaf removal might negate the

signal for the plant to respond (Walters 2010). Some small mammals, such as voles

selectively remove the basal meristems of grasses and may disrupt the cell walls, whereas

larger herbivores, such as ungulates, remove large portions of the above ground biomass in a

single bite, a very different type of tissue wounding. There are few studies addressing this,

though Massey et al. (2007b) compared silicon induction in response to mechanical damage

and herbivory by locusts and voles. They demonstrated that although both types of herbivory

induced silicon defences more than clipping, there was no difference between the impacts of

the two herbivores on two different natural grasses.

Despite the tendency for insect and mammalian herbivores to elicit induction of silicon

defences, this pattern is not universal; some studies have found that herbivory did not cause a

measureable induction of silicon defences (e.g. Soininen et al. 2013; Quigley & Anderson

2014). These examples tend to be field-based studies comparing silicon levels in grasses in

grazed and ungrazed areas, where the levels of herbivory are unknown and maybe of

insufficient duration and/or intensity to elicit induction (see below), and where other site-

based factors, e.g. local climate, soil type, or previous grazing history, may influence

induction (e.g. Georgiadis & McNaughton 1990; Fenner, Lee & Duncan 1993; Soininen et al.

2013). Laboratory studies may provide an explanation as some have demonstrated that

silicon induction may require a threshold of damage, either in terms of amount of biomass

removed or in terms of frequency of damage (Massey, Ennos & Hartley 2007b; Reynolds et

9

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

al. 2012). These studies suggest a single instance of damage does not lead to induction, nor

do damage levels of less than around 20% of total leaf area removed.

Case study: The effects of grazing by voles on silicon induction in the field

The complexity of the relationship between induction and damage intensity has been difficult

to resolve given the lack of studies in the field; clear thresholds of herbivore damage required

to induce elevated silica accumulation have only been demonstrated in laboratory studies.

Recently, we conducted field experiments using specially-constructed grazing enclosures

which exposed Deschampsia caespitosa plants to varying intensities of grazing by field voles

(Microtus agrestis) to test the effects of grazing intensity and season on silicon induction (J.

DeGabriel, S. Hartley, F. Massey, S. Reidinger and X. Lambin, unpublished data). We

compared our field results to the laboratory results of Reynolds et al. (2012), using the same

study system.

Methods

Experimental design

We erected a 36 m x 36 m grazing enclosure, consisting of 81 4 m x 4 m cells in an area of

natural clear cut grassland in Kielder forest in northern England that is habitat for populations

of field voles. The enclosures were constructed from vole-proof wire mesh, which was

sunken 30 cm below ground and was at least 50 cm high, topped with a roll-top, which

prevented voles from moving into neighbouring cells. The dominant plant species in each of

the experimental cells was D. caespitosa, which is a major dietary component of field voles

and their main overwinter food source. The enclosures were exposed to natural levels of vole

grazing in previous years, but we trapped and removed all voles from the enclosures in the

winter before commencing our experiment in spring.

10

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

From March 2009, we live-trapped wild voles in surrounding grassland using Ugglan traps

(Grahnab, Marieholm, Sweden) and immediately introduced a single vole into each of 12

cells (giving a density of 50 voles/ha) and 6 voles into each of another 12 cells (giving a

density of 300 voles/ha). The sex and body mass of each vole was recorded. Voles were

allowed to graze freely in the cells for 3-4 days, before we re-trapped and released them

outside the enclosures. We repeated this grazing treatment roughly every six-seven weeks

between March and November 2009, as well as in January, February and April 2010. Ability

to access field sites over winter was restricted due to heavy snow.

We collected samples from a single D. caespitosa tussock in each enclosure approximately

one month after each grazing treatment. Within each cell, we randomly chose 3 tussocks on

each sampling occasion and took 5 tillers each from the centre and edge of those tussocks.

We pooled the leaves from the three plants in plastic bags and stored them frozen at -20°C for

analysis. The leaves chosen were the youngest fully expanded and undamaged adult leaf

blades available that were green and not contaminated with fungus, which we considered to

be the most palatable to voles. Thus, at different times of year, the leaf samples were not

exactly the same, as we deliberately did not collect new or young leaves that had not fully

matured. We prepared and analysed the silicon content of the leaf samples using portable X-

Ray Fluorescence (Reidinger, Ramsey & Hartley 2012).

In September 2009, we estimated the average grazing damage levels on D. caespitosa. We

randomly selected a single tussock in each cell and haphazardly chose 100 leaves on the

outside of the plant (covering the entire circumference of the tussock) and 100 leaves on the

11

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

interior. We visually recorded how many of these leaves had been damaged by vole grazing

and averaged the proportion of leaves damaged across the plant.

Results

Effects of grazing intensity on silicon induction

We found that on average, approximately 5 % of leaves were damaged in the 50 voles/ha

treatment and 23.5 % of leaves were damaged in the 300 voles/ha treatment. This was

roughly equivalent to the “low” (5% of leaves removed) and “high” (20% of leaves removed)

grazing treatments imposed in the laboratory study by Reynolds et al. (2012). We found

remarkably similar patterns in the rates of silicon induction under the high and low grazing

pressures in the field (Figure 1a) to those reported by Reynolds et al. (2012). In both the lab

and the field, silicon induction only occurred under the high grazing intensity treatment, but

not the low. Furthermore, induction was delayed for two months after initiation of grazing,

before an approximate doubling of silicon concentrations in the high, relative to the low

grazing treatment by five months after the start of damage.

Effects of season on silicon induction

We found that silicon concentrations increased in D. caespitosa in response to vole grazing

during the summer and autumn, reaching a peak in winter, presumably as a result of

accumulation in old leaves from the previous growing season. Concentrations of silicon then

decreased rapidly in the spring, again presumably as a result of flushes of new leaves that had

not taken up silicon (Figure 1b).

Our results demonstrate that both threshold effects and seasonality are important in silicon

uptake, and these factors have been found to influence induction in other studies. For

12

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

example, in a study of Agrostis tenuis, Banuelos and Obeso (2000) found that silicon content

of plants was higher in heavily grazed areas than within experimental exclosures during the

summer, but no such differences were apparent in winter. This was in contrast to the results

from our experimental field enclosures in northern England (Figure 1b). There are similar

phenological effects in plant responses to clipping: in a study of 5 tropical grass species,

silicon content generally increased over time, although this effect varied with species, and for

some species the effect of clipping on leaf sheath silicon content differed between dates

(Kindomihou, Sinsin & Meerts 2006). Similarly, the effect of mowing on the silicon levels of

prairie foliage differed between July, when there was no effect, and October, when there was

an increase (Seastedt, Ramundo & Hayes 1989).

There is also evidence that phenological variation in silicon content may differ between grass

species growing in different locations. For example, in North American prairies shoot silicon

concentrations increase throughout the growing season (Brizuela, Detling & Cid 1986;

Seastedt, Ramundo & Hayes 1989), and the same trend was found in savanna grasses in

Kenya (Georgiadis & McNaughton 1990). In contrast, in another African study, grasses in the

Serengeti in Tanzania, had higher silicon levels early in the growing season (McNaughton et

al. 1985). This variation is more likely related to broader ecosystem differences across

latitudes than effects of season per se. Clearly induction of silicon defences, whether in

response to artificial damage or natural herbivory is highly variable and its magnitude is

contingent on a number of factors, including damage type, damage intensity, timing of

damage, plant species and even tissue age (see below, Banuelos & Obeso 2000; Kindomihou,

Sinsin & Meerts 2006).

13

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

Impact of herbivory: induction of silicon defences varies with plant species

and genotype

The ability of different non-agricultural grass species to increase their silicon uptake in

response to experimental removal of leaves by herbivores has been measured across a

relatively narrow range of species under controlled conditions (Massey, Ennos & Hartley

2006; Massey, Ennos & Hartley 2007b). In contrast, many studies have assessed such

variability in relation to clipping and have demonstrated clear between-species differences in

silicon uptake in response (e.g. McNaughton et al. 1985; Kindomihou, Sinsin & Meerts 2006;

Soininen et al. 2013). Between-species variation in the magnitude of the differences in silicon

levels in wild plants collected from naturally grazed versus ungrazed areas is also well-

documented (e.g. McNaughton & Tarrants 1983; Brizuela, Detling & Cid 1986; Soininen et

al. 2013). Such differences have also been demonstrated within-species, which has led to the

suggestion that herbivory drives the selection of ecotypes with increased ability to take up

silicon (Detling & Painter 1983; McNaughton & Tarrants 1983; Banuelos & Obeso 2000).

While that idea remains somewhat speculative for field populations, the existence of intra-

specific genotypic differences in silicon induction in response to clipping is clear in

laboratory experiments. For example, the silicon content of some genotypes of A. tenuis

increased after clipping, whereas it declined in others (Banuelos & Obeso 2000). Similarly,

Soininen et al. (2013) found that four different grass species showed marked within-species

differences in silicon content following clipping, in addition to extensive between-species

variation. Similarly, three grass species from the same genus, Festuca, showed very different

patterns of silicon uptake and deposition in defensive structures (spines and phytoliths) in

response to artificial damage and manipulation of silicon supply, as did two genotypes of one

of these species, F. arundinacea (Hartley et al. 2015).

14

320

321

322

323

324

325

326

327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

We do not know why there are such large differences in silicon content in quite closely

related species and even between genotypes of the same species. This is because we have a

very limited understanding of silicon uptake at the physiological, biochemical and molecular

level for most non-crop species (Hartley et al. 2015; though see Deshmukh & Belanger

2016). In crop species, particularly rice, many of the transporters responsible for silicon

uptake and distribution within the plant have been identified and their role characterised (Ma

et al. 2006; Ma & Yamaji 2006; Ma 2009; Ma & Yamaji 2015; Yamaji et al. 2015), but we

still have limited understanding of how the impact of damage on silicon uptake and

deposition interacts with abiotic factors. In addition, grasses have an array of different types

of defences, which is a complicating factor with respect to disentangling silicon dynamics.

Thus far, few studies (but see below Quigley & Anderson 2014; Wieczorek et al. 2015b)

have attempted to simultaneously quantify experimentally the relative importance of biotic

factors, such as grazing or other grass defences, and abiotic factors, such as water availability,

on silicon uptake, particularly in the field.

Abiotic factors: induction of silicon defences in response to herbivory

varies with soil type, water availability, and climate

Abiotic factors influence silicon levels and can impact silicon defences (Soininen et al.

2013), although in many studies it is hard to disentangle abiotic from biotic influences,

particularly grazing levels. For example, it is unclear whether higher levels of silicon

observed in plants from grazed sites in the North American prairies (Brizuela, Detling & Cid

1986) or the Serengeti (McNaughton et al. 1985) are due to a direct response to herbivory

(i.e. induction), or to other abiotic differences between the sites. However, it is clear that

plants from more heavily grazed sites could accumulate more silicon in leaves than those

from ungrazed ones in the laboratory (Detling & Painter 1983; McNaughton & Tarrants

15

345

346

347

348

349

350

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

1983), suggesting some role for biotic drivers, regardless of abiotic conditions. However,

uptake ultimately depends on availability of silicon, itself dependent on soil type and soil pH

(Beckwith & Reeve 1964; Ehrlich et al. 2010) and, since silicon enters the plants in soluble

form through the transpiration stream, it may also depend on water availability and climatic

factors which influence transpiration, such as temperature (e.g. Raven 1983; Sangster,

Hodson & Tubb 2001; Kindomihou, Sinsin & Meerts 2006; Faisal et al. 2012). However, the

extent to which silicon uptake depends on transpiration rate remains a subject of debate

(Hartley et al. 2015).

A recent study by Wieczorek et al. (2015b) attempted to disentangle the relative importance

of abiotic and biotic factors in silicon accumulation in a natural wetland system, where the

dynamic hydrology might be predicted to have as large an impact as herbivory on the silicon

content of foliage. They demonstrated the importance of abiotic factors in silicon

accumulation in grazed systems, with temperature and snow cover influencing silicon uptake

in both leaves and rhizomes of a tussock sedge, whilst the level of winter flooding affected

uptake in the rhizomes, but not in the leaves. However, although both herbivory and abiotic

conditions influenced the uptake of soil available silicon by plants in this study, grazing

appeared to be a more important driver than hydrology for foliar tissue (Wieczorek et al.

2015b). This contrasts with the study by Quigley and Anderson (2014), which found water

availability had a greater impact on natural silicon levels than defoliation in one of the two

species tested, although this study used clipping rather than natural herbivory.

Abiotic and biotic factors may interact in determining both the levels of silicon-based

defences and their impact on herbivores. For example, the effectiveness of silicon-based plant

defences against locusts has been shown to differ between plant species according to soil

16

370

371

372

373

374

375

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

silicon availability. Under low soil silicon availability, the herbivores removed more leaf

biomass from L. perenne than from P. annua, whereas under high silicon availability the

reverse was true. Consequently, herbivory shifted the competitive balance between the two

grass species, with the outcome depending on the availability of soil silicon (Garbuzov,

Reidinger & Hartley 2011). Overall, we see evidence that abiotic factors influence silicon-

based responses to damage in plants, but we currently lack comprehensive experimental

evidence of these influences, particularly in the case of field studies involving herbivores.

Interactions between environmental drivers such as soil silicon and water availability and

induction of silicon uptake in response to damage appear to be complex (Kindomihou, Sinsin

& Meerts 2006; Soininen et al. 2013; Quigley & Anderson 2014).

Impacts of silicon defences on herbivores vary with herbivore type

Ecological studies with invertebrates feeding on natural grasses have demonstrated strong

negative effects of plant silicon uptake on rates of herbivory and larval growth rates in a

range of species across various feeding guilds (Massey, Ennos & Hartley 2006; Massey,

Ennos & Hartley 2007b; Massey & Hartley 2009). However, to date many studies with

invertebrates have been in crop species (e.g. Goussain, Prado & Moraes 2005; Kvedaras &

Keeping 2007; Kvedaras et al. 2007; Kvedaras et al. 2009; Griffin, Hogan & Schmidt 2015)

and some have involved measuring effects when silicon has been sprayed on the plant

surface, rather than being taken up and deposited naturally by the plant, which is likely to

impact on herbivore responses (Moraes et al. 2004; Eswaran & Manivannan 2007).

Ecological studies on the impacts of silicon on herbivores below-ground are particularly

lacking. In one of the very few studies on this topic, silicon addition had no effect on root

herbivores (masked chafer grubs), despite causing an increase in both root and leaf silicon

content (Redmond & Potter 2006).

17

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416

417

418

419

Similarly, only a relatively small number of studies have investigated the impacts of silicon

defences on the food preferences and performance of mammals, but there is some evidence

emerging which suggests silicon has a greater impact on the feeding behaviour of smaller

herbivores, compared to larger species. For example, laboratory studies with captive animals

have convincingly demonstrated that field voles, prairie voles (M. ochrogaster) and rabbits

(Oryctolagus cuniculus) consistently reduce their consumption of grass species containing

high concentrations of silicon (Gali-Muhtasib, Smith & Higgins 1992; Massey & Hartley

2006; Cotterill et al. 2007). Furthermore, field voles fed diets containing higher

concentrations of silicon exhibited slower growth rates and higher mortality under controlled

conditions (Massey & Hartley 2006; Huitu et al. 2014). In contrast, Massey et al. (2009)

found that sheep were less impacted by silicon defences than were smaller herbivores,

although more studies on larger grazers are required to confirm the consistency of this

pattern.

One possible reason for observed differences in effects of silicon in grasses on insects and

larger mammalian herbivores may be attributed to the differential impacts of the wearing of

teeth and mouthparts (reviewed by Strömberg, Di Stilio & Song 2016). Silicon phytoliths

have been clearly shown to cause significant and irreversible mandibular wear in the

lepidopteran Spodoptera (Massey & Hartley 2009; Reynolds, Keeping & Meyer 2009); the

extent of wear correlated with a reduction in digestive efficiency of the caterpillars,

suggesting that such wear could contribute to diet selection and the impact of silicon on

herbivore growth rates (Massey & Hartley 2009). In addition, the extent and nature of

deposition of silicon at the leaf surface has been shown to influence the abrasiveness of

natural grass species and hence potentially their vulnerability to herbivores (Hartley et al.

18

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

435

436

437

438

439

440

441

442

443

444

2015). In contrast, recent studies have demonstrated that silicon phytoliths in many grass

species are softer than tooth enamel of mammal groups including ungulates, macropods and

primates (Sanson, Kerr & Gross 2007; Rabenold & Pearson 2011; Erickson 2014; Lucas et

al. 2014; Rivals et al. 2014). However, there is evidence that some grass species contain

phytoliths that are harder than tooth enamel (Erickson 2014), although whether these are

selected by herbivores is unclear. Furthermore, Calandra et al. (2016) found effects of silicon

on microwear patterns in the teeth of voles and have proposed this as a mechanism by which

silicon may contribute to population crashes. Hummel et al. (2011), provide a compelling

argument for a role of silicon in the evolution of high-crowned teeth, showing a strong

positive correlation between faecal silicon levels and hypsodonty across a range of large

African herbivores with differing diets and digestive systems. McArthur (2014) points out

that teeth and chewing are an often neglected, but crucial component of understanding

herbivore diet selection, especially given the importance of food processing time on

digestion. In support of this idea, high silicon levels have been shown to reduce the bite rate

of sheep (Massey et al. 2009), with impacts on processing time and digestive efficiency likely

to explain why the sheep preferred to feed on grasses low in silicon.

It has been suggested that while phytoliths may not wear down mammalian teeth, they may

reduce animals’ access to cell contents by preventing cell walls being broken apart (Massey

& Hartley 2006). Consequently, variation in age, body size and digestive physiology may

play a role in determining differential effects of silicon. Variation in bite size and offtake rate

among different size classes of herbivores may impact the induction of silicon defences,

whilst the greater amount of biomass ingested by large herbivores could potentially dilute the

potency of silicon defence. Negative relationships between herbivore body size and diet

quality as a result of increased digestive efficiency have been well described (Bell 1970;

19

445

446

447

448

449

450

451

452

453

454

455

456

457

458

459

460

461

462

463

464

465

466

467

468

469

Jarman 1974), although a recent paper by Steuer et al. (2014) challenges this paradigm.

Research to date has generally focussed on the positive aspects of animal nutrition, but an

understanding of the interactive role of plant defences on the digestibility of plants for

different size and age classes of herbivores is missing. Silicon defences in grasses are an

excellent system to test such nutritional hypotheses.

Most grazers have developed the ability to digest a lot of fibre in grasses, but not silicon.

Thus, it may act as an effective bulking agent and prevent fibre (structural carbohydrates),

and ultimately, dry matter digestibility (Shewmaker et al. 1989). As epidermal silicon can

prevent enzyme-aided infiltration by fungal hyphae (Fauteux et al. 2006), it seems likely that

it can protect some of the fibre fractions from degradation by cellulases. Watling et al. (2011)

found that carbon occluded in phytoliths includes cellulose, lignin and carboxylic acids,

which suggests that there could be some chemical interaction between these fractions. In

addition, silicon is likely to impact on nitrogen absorption by preventing the leaf cell walls

being broken apart (Massey & Hartley 2006; Hunt et al. 2008), which is presumably one way

silicon reduces growth rates and fecundity of voles and insects. The impact is predicted to be

more marked in small, hindgut-fermenting herbivores, such as voles which are more likely to

be N limited, compared to the larger ruminants which can utilise endogenous sources of N, or

lagomorphs which practice caecotrophy to avoid N limitation. Nevertheless, silicon has been

shown to inhibit microbial digestion in ruminants (Harbers, Raiten & Paulsen 1981), so

further studies are required to validate this hypothesis. Wieczorek et al. (2015a) elucidated

the physiological mechanisms underpinning the negative effects of an abrasive plant diet on

the performance of root voles (M. oeconomus). Voles fed a diet of sedges containing silicon

and high concentrations of fibre had reduced absorptive efficiency in the small intestine, with

shorter villi and more mucus cells, compared to controls. Consequently, these animals had

20

470

471

472

473

474

475

476

477

478

479

480

481

482

483

484

485

486

487

488

489

490

491

492

493

494

reduced body mass and lower resting metabolic rate, which they suggested was because voles

were unable to increase food intake sufficiently to compensate for the impacts of

abrasiveness on the gut. Further studies on the physiological impact of silicon abrasiveness

on vertebrate guts would be intriguing.

The impacts of silicon on herbivore growth rates and reproduction are predicted to be more

significant for herbivores that exhibit population cycles, such as voles (Reynolds et al. 2012),

since the negative feedback from delayed density-dependence of silicon induction in relation

to herbivore density provides a nutritional mechanism to explain population regulation

(Massey & Hartley 2006; Massey et al. 2008; Wieczorek et al. 2015b). Conversely, feedback

between herbivore population density and grazing pressure means that cyclic herbivore

species are more likely to drive patterns of silicon induction, compared to non-cyclic

herbivores (Wieczorek et al. 2015b). Theoretical models have provided support for this

hypothesis, indicating that a threshold level of herbivore damage is required to initiate

sufficient silicon induction to elicit population cycles (Reynolds et al. 2012). Recently,

Wieczorek et al. (2015b) demonstrated that grazing by voles at a spatial scale relevant to their

home ranges resulted in significant induction of silicon defences in sedges in Poland, while

Massey et al. (2008) found correlations between silicon levels in D. caespitosa and M.

agrestis densities in northern England. However, as yet there have been no empirical studies

in natural grasslands at the landscape scale relevant to animal populations which

convincingly demonstrate that vole grazing pressure is sufficient to induce silicon defences to

the level required to affect herbivore population dynamics (Hartley 2015). Nevertheless, the

work in Polish and English grassland systems, including the advances in understanding the

effects of eating high-silicon diets on animals’ digestive physiology (Wieczorek et al. 2015a),

gives some support to the hypothesis that silicon defences may drive vole population cycles.

21

495

496

497

498

499

500

501

502

503

504

505

506

507

508

509

510

511

512

513

514

515

516

517

518

519

The next step is to expand these studies to understand how local effects of silicon on vole

meta-populations drive population cycles at a landscape scale.

Landscape scale studies of wild herbivore populations

Linking plant defence to the regulation of wild herbivore populations is inherently difficult

(Bazely et al. 1997; Foley, Iason & Makkar 2007; DeGabriel et al. 2014). Two studies have

successfully demonstrated relationships between N availability and reproductive success in

mammal populations mediated by constitutive tannin concentrations (DeGabriel et al. 2009;

McArt et al. 2009), but no such relationships have as yet been demonstrated for induced

defence systems. Attempts to link silicon defences to mammal population cycles are

hampered by the complexity of the diets of wild herbivores in natural grasslands, which may

result in insufficient grazing pressure on a single plant species to induce silicon to levels

comparable to those producing anti-herbivore effects in no-choice laboratory studies.

Secondly, spatial variation in silicon concentrations as a result of the biotic and abiotic

factors described above means that averaged values for a site may under- or over-estimate the

extreme values that animals ingest within their home ranges. Finally, at certain time points,

e.g. during the “crash phase” of a cycle, natural grazing intensity may be insufficient to elicit

high levels of silicon induction. These effects are evident from Figure 1a as, despite the

similarities in patterns of silicon induction between the laboratory and field studies, the

absolute concentrations of silicon in the plants grown in the glasshouse were significantly

higher than the plants from the field. Given the complexities of the field environment, to

reveal relationships between induction of silicon defences and herbivore population dynamics

in natural grasslands we need to first obtain quantitative data on the intensity of grazing on

individual grass species in order to have confidence that herbivores are eating silicon-

accumulating plants. We also need to design sampling strategies with sufficient numbers of

22

520

521

522

523

524

525

526

527

528

529

530

531

532

533

534

535

536

537

538

539

540

541

542

543

544

samples collected across an appropriate spatial scale to capture the variation in silicon

concentrations in field environments. We need to select places and times where herbivore

densities are high enough to elicit sufficient grazing pressure to exceed the threshold required

to cause induction of silicon defences. Finally, we need to be aware of abiotic factors that

may impact on silicon induction, as described above, and use this information to inform our

selection of sites and the timing of our experimental manipulations and sample collection.

Conclusion

Much is now known about silicon-based defences in grasses and their impact on herbivores

(Figure 2), though it is also clear that silicon defences in natural grasses exhibit enormous

variability, both within and between species. Induction of silicon defences is affected by

abiotic factors such as soil silicon availability, by variation in biological process such as

transpiration rates, and by plant genotype, as well as by the amount and type of damage a

plant receives (Figure 2). However, much of this current understanding has been derived

from studies in the laboratory and glasshouse, which is in large part due to difficulties

inherent in field studies, where multiple, interacting factors may simultaneously impact on

the uptake and use of silicon for defence. Although relationships between silicon

concentration and animal feeding preferences and performance can be demonstrated in the

laboratory (Massey & Hartley 2006; Massey, Ennos & Hartley 2007b), as we increase spatial

scale the effects of grazing on silica induction become harder to demonstrate, particularly at a

landscape scale (Soininen et al. 2013; Huitu et al. 2014; Wieczorek et al. 2015b). Only a

handful of large-scale studies have been conducted so far, but encouragingly, the patterns of

induction, in terms of threshold damage levels required, time for it to occur and its magnitude

seem similar in the glasshouse and in enclosures (Figure 1). Crucially though, we still lack a

landscape scale demonstration of the impact of herbivores on silicon induction and vice

23

545

546

547

548

549

550

551

552

553

554

555

556

557

558

559

560

561

562

563

564

565

566

567

568

569

versa. This does not indicate that silicon defences do not have any functional relevance in real

ecosystems. Rather, ecologists need to overcome the difficulties inherent in observing effects

at landscape scales where there is a need to tease apart the confounding factors that could

impact silicon induction and its effect on herbivores. There are a number of other such key

knowledge gaps which currently prevent us having a full understanding of the ecological role

of silicon-based defences against herbivores. We highlight some of them in Figure 2 and

suggest them as potential future research areas to provide novel insights into the mechanisms

by which silicon can underpin plant-herbivore interactions in grasses.

Acknowledgements

We thank Julia Cooke, Ben Moore and anonymous referees for comments on earlier drafts.

Xavier Lambin, Fergus Massey, Stefan Reidinger and Elizabeth Herridge contributed to the

unpublished study described in Figure 1, which was funded by NERC grants to SEH and XL

((NE/F003137/1 and NE/F003994/1).

24

570

571

572

573

574

575

576

577

578

579

580

581

582

583

References

Alborn, H.T., Turlings, T.C.J., Jones, T.H., Stenhagen, G., Loughrin, J.H. & Tumlinson, J.H.

(1997) An elicitor of plant volatiles from beet armyworm oral secretion. Science, 276,

945-949.

Banuelos, M.J. & Obeso, J.R. (2000) Effect of grazing history, experimental defoliation, and

genotype on patterns of silicification in Agrostis tenuis Sibth. Ecoscience, 7, 45-50.

Bazely, D.R., Vicari, M., Emmerich, S., Filip, L., Lin, D. & Inman, A. (1997) Interactions

between herbivores and endophyte-infected Festuca rubra from the Scottish islands of

St. Kilda, Benbecula and Rum. Journal of Applied Ecology, 34, 847-860.

Beckwith, R.S. & Reeve, R. (1964) Studies on soluble silica in soils. II. The release of

monosilicic acid from soils. Australian Journal of Soil Research, 2, 157-168.

Bell, R.H.V. (1970) The use of the herb layer by grazing ungulates in the Serengeti. Animal

Populations and Relations to their Food Resources (ed. A. Watson), pp. 111-124.

Blackwell, Oxford.

Bonaventure, G. (2012) Perception of insect feeding by plants. Plant Biology, 14, 872-880.

Bonaventure, G., VanDoorn, A. & Baldwin, I.T. (2011) Herbivore-associated elicitors: FAC

signaling and metabolism. Trends in Plant Science, 16, 294-299.

Brizuela, M.A., Detling, J.K. & Cid, M.S. (1986) Silicon concentration of grasses growing in

sites with different grazing histories. Ecology, 67, 1098-1101.

Calandra, I., Zub, K., Szafranska, P.A., Zalewski, A. & Merceron, G. (2016) Silicon-based

plant defences, tooth wear and voles. Journal of Experimental Biology, 219, 501-507.

Cid, M.S., Detling, J.K., Brizuela, M.A. & Whicker, A.D. (1989) Patterns in grass

silicification: response to grazing history and defoliation. Oecologia, 80, 268-271.

25

584

585

586

587

588

589

590

591

592

593

594

595

596

597

598

599

600

601

602

603

604

605

606

Cid, M.S., Detling, J.K., Whicker, A.D. & Brizuela, M.A. (1990) Silicon uptake and

distribution in Agropyron smithii as related to grazing history and defoliation. Journal

of Range Management, 43, 344-346.

Cooke, J. & Leishman, M.R. (2011) Is plant ecology more siliceous than we realise? Trends

in Plant Science, 16, 61-68.

Cotterill, J.V., Watkins, R.W., Brennon, C.B. & Cowan, D.P. (2007) Boosting silica levels in

wheat leaves reduces grazing by rabbits. Pest Management Science, 63, 247-253.

Currie, H.A. & Perry, C.C. (2007) Silica in plants: Biological, biochemical and chemical

studies. Annals of Botany, 100, 1383-1389.

Damuth, J. & Janis, C.M. (2011) On the relationship between hypsodonty and feeding

ecology in ungulate mammals, and its utility in palaeoecology. Biological Reviews,

86, 733-758.

DeGabriel, J.L., Moore, B.D., Felton, A.M., Ganzhorn, J.U., Stolter, C., Wallis, I.R.,

Johnson, C.N. & Foley, W.J. (2014) Translating nutritional ecology from the

laboratory to the field: milestones in linking plant chemistry to population regulation

in mammalian browsers. Oikos, 123, 298-308.

DeGabriel, J.L., Moore, B.D., Foley, W.J. & Johnson, C.N. (2009) The effects of plant

defensive chemistry on nutrient availability predict reproductive success in a

mammal. Ecology, 90, 711-719.

Deshmukh, R.K. & Belanger, R.R. (2016) Molecular evolution of aquaporins and silicon

influx in plants. Functional Ecology, In press.

Detling, J.K. & Painter, E.L. (1983) Defoliation responses of western wheatgrass populations

with diverse histories of prarie dog grazing. Oecologia, 57, 65-71.

26

607

608

609

610

611

612

613

614

615

616

617

618

619

620

621

622

623

624

625

626

627

628

629

Ehrlich, H., Demadis, K.D., Pokrovsky, O.S. & Koutsoukos, P.G. (2010) Modern Views on

Desilicification: Biosilica and Abiotic Silica Dissolution in Natural and Artificial

Environments. Chemical Reviews, 110, 4656-4689.

Epstein, E. (1999) Silicon. Annual Review of Plant Physiology and Plant Molecular Biology,

50, 641-664.

Ergon, T., Ergon, R., Begon, M., Telfer, S. & Lambin, X. (2011) Delayed density-dependent

onset of spring reproduction in a fluctuating population of field voles. Oikos, 120,

934-940.

Ergon, T., Lambin, X. & Stenseth, N.C. (2001) Life-history traits of voles in a fluctuating

population respond to the immediate environment. Nature, 411, 1043-1045.

Erickson, K.L. (2014) Prairie grass phytolith hardness and the evolution of ungulate

hypsodonty. Historical Biology, 26, 737-744.

Eswaran, A. & Manivannan, K. (2007) Effect of foliar application of lignite fly ash on the

management of papaya leaf curl disease. Acta Horticulturae, 740, 271-275.

Faisal, S., Callis, K.L., Slot, M. & Kitajima, K. (2012) Transpiration-dependent passive silica

accumulation in cucumber (Cucumis sativus) under varying soil silicon availability.

Botany-Botanique, 90, 1058-1064.

Farmer, E. (2014) Leaf Defence. Oxford University Press, Oxford, U.K.

Fauteux, F., Chain, F., Belzile, F., Menzies, J.G. & Belanger, R.R. (2006) The protective role

of silicon in the Arabidopsis-powdery mildew pathosystem. Proceedings of the

National Academy of Sciences of the United States of America, 103, 17554-17559.

Fenner, M., Lee, W.G. & Duncan, S.J. (1993) Chemical features of Chionochloa species in

relation to grazing by ruminants in South Island, New Zealand. New Zealand Journal

of Ecology, 17, 35-40.

27

630

631

632

633

634

635

636

637

638

639

640

641

642

643

644

645

646

647

648

649

650

651

652

653

Foley, W., Iason, G. & Makkar, H. (2007) Transdisciplinary studies of plant secondary

metabolites: Lessons from ecology for animal science and vice versa. Proceedings of

the 7th International Symposium on the Nutrition of Herbivores (eds Q.X. Meng, J.X.

Liu & W.Y. Zhu). Beijing.

Gali-Muhtasib, H.U., Smith, C.C. & Higgins, J.J. (1992) The effect of silica in grasses on the

feeding behavior of the prairie vole Microtus ochrogaster. Ecology, 73, 1724-1729.

Garbuzov, M., Reidinger, S. & Hartley, S.E. (2011) Interactive effects of plant-available soil

silicon and herbivory on competition between two grass species. Annals of Botany,

108, 1355-1363.

Georgiadis, N.J. & McNaughton, S.J. (1990) Elemental and fiber contents of savanna grasses:

variation with grazing, soil type, season and species. Journal of Applied Ecology, 27,

623-634.

Gomes, F.B., de Moraes, J.C., dos Santos, C.D. & Goussain, M.M. (2005) Resistance

induction in wheat plants by silicon and aphids. Scientia Agricola, 62, 547-551.

Goussain, M.M., Prado, E. & Moraes, J.C. (2005) Effect of silicon applied to wheat plants on

the biology and probing behaviour of the greenbug Schizaphis graminum (Rond.)

(Hemiptera : Aphididae). Neotropical Entomology, 34, 807-813.

Griffin, M., Hogan, B. & Schmidt, O. (2015) Silicon reduces slug feeding on wheat

seedlings. Journal of Pest Science, 88, 17-24.

Harbers, L.H., Raiten, D.J. & Paulsen, G.M. (1981) The role of plant epidermal silica as a

structural inhibitor of rumen microbial digestion in steers. Nutrition Reports

International, 24, 1057-1066.

Harbers, L.H. & Thouvenelle, M.L. (1980) Digestion of corn and sorghum silage observed by

scanning electron microscopy. Journal of Animal Science, 50, 514-526.

28

654

655

656

657

658

659

660

661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

Hartley, S.E. (2015) Round and round in cycles? Silicon-based plant defences and vole

population dynamics. Functional Ecology, 29, 151-153.

Hartley, S.E., Fitt, R.N., McLamon, E.L. & Wade, R.N. (2015) Defending the leaf surface:

intra- and inter-specific differences in silicon deposition in grasses in response to

damage and silicon supply. Frontiers in Plant Science, 6, 35.

Hartley, S.E. & Lawton, J.H. (1987) Effects of different types of damage on the chemistry of

birch foliage, and the responses of birch feeding insects. Oecologia, 74, 432-437.

Hartley, S.E. & Lawton, J.H. (1991) Biochemical aspects and significance of the rapidly

induced accumulation of phenolics in birch foliage. Phytochemical Induction by

Herbivores (eds D.W. Tallamy & M.J. Raupp), pp. 105-132. Wiley, New York.

Haukioja, E. & Neuvonen, S. (1985) Induced long-term resistance of birch foliage against

defoliators - defensive or incidental? Ecology, 66, 1303-1308.

Heil, M. & Ton, J. (2008) Long-distance signalling in plant defence. Trends in Plant Science,

13, 264-272.

Hodson, M.J., White, P.J., Mead, A. & Broadley, M.R. (2005) Phylogenetic variation in the

silicon composition of plants. Annals of Botany, 96, 1027-1046.

Huitu, O., Forbes, K.M., Helander, M., Julkunen-Tiitto, R., Lambin, X., Saikkonen, K.,

Stuart, P., Sulkama, S. & Hartley, S. (2014) Silicon, endophytes and secondary

metabolites as grass defenses against mammalian herbivores. Frontiers in Plant

Science, 5, 478.

Hummel, J., Findeisen, E., Suedekum, K.-H., Ruf, I., Kaiser, T.M., Bucher, M., Clauss, M. &

Codron, D. (2011) Another one bites the dust: faecal silica levels in large herbivores

correlate with high-crowned teeth. Proceedings of the Royal Society B-Biological

Sciences, 278, 1742-1747.

29

678

679

680

681

682

683

684

685

686

687

688

689

690

691

692

693

694

695

696

697

698

699

700

701

Hunt, J.W., Dean, A.P., Webster, R.E., Johnson, G.N. & Ennos, A.R. (2008) A novel

mechanism by which silica defends grasses against herbivory. Annals of Botany, 102,

653-656.

Jarman, P.J. (1974) Social organization of antelope in relation to their ecology. Behaviour,

48, 215-267.

Karban, R. & Baldwin, I.T. (1997) Induced Responses to Herbivory. The University of

Chicago Press, Chicago.

Katz, O. (2015) Silica phytoliths in angiosperms: phylogeny and early evolutionary history.

New Phytologist, 208, 642-646.

Keeping, M.G., Meyer, J.H. & Sewpersad, C. (2013) Soil silicon amendments increase

resistance of sugarcane to stalk borer Eldana saccharina Walker (Lepidoptera:

Pyralidae) under field conditions. Plant and Soil, 363, 297-318.

Keeping, M.G., Miles, N. & Sewpersad, C. (2014) Silicon reduces impact of plant nitrogen in

promoting stalk borer (Eldana saccharina) but not sugarcane thrips (Fulmekiola

serrata) infestations in sugarcane. Frontiers in Plant Science, 5, 289.

Keeping, M.G. & Reynolds, O.L. (2009) Silicon in agriculture: new insights, new

significance and growing application. Annals of Applied Biology, 155, 153-154.

Kindomihou, V., Sinsin, B. & Meerts, P. (2006) Effect of defoliation on silica accumulation

in five tropical fodder grass species in Benin. Belgian Journal of Botany, 139, 87-102.

Korth, K.L. & Dixon, R.A. (1997) Evidence for chewing insect-specific molecular events

distinct from a general wound response in leaves. Plant Physiology, 115, 1299-1305.

Kvedaras, O.L., Byrne, M.J., Coombes, N.E. & Keeping, M.G. (2009) Influence of plant

silicon and sugarcane cultivar on mandibular wear in the stalk borer Eldana

saccharina. Agricultural and Forest Entomology, 11, 301-306.

30

702

703

704

705

706

707

708

709

710

711

712

713

714

715

716

717

718

719

720

721

722

723

724

725

Kvedaras, O.L. & Keeping, M.G. (2007) Silicon impedes stalk penetration by the borer

Eldana saccharina in sugarcane. Entomologia Experimentalis Et Applicata, 125, 103-

110.

Kvedaras, O.L., Keeping, M.G., Goebel, F.R. & Byrne, M.J. (2007) Larval performance of

the pyralid borer Eldana saccharina Walker and stalk damage in sugarcane: Influence

of plant silicon, cultivar and feeding site. International Journal of Pest Management,

53, 183-194.

Lindroth, R.L. & Batzli, G.O. (1986) Inducible plant chemical defences - A cause of vole

population cycles. Journal of Animal Ecology, 55, 431-449.

Lucas, P.W., van Casteren, A., Al-Fadhalah, K., Almusallam, A.S., Henry, A.G., Michael, S.,

Watzke, J., Reed, D.A., Diekwisch, T.G.H., Strait, D.S. & Atkins, A.G. (2014) The

role of dust, grit and phytoliths in tooth wear. Annales Zoologici Fennici, 51, 143-152.

Ma, J.F. (2004) Role of silicon in enhancing the resistance of plants to biotic and abiotic

stresses. Soil Science and Plant Nutrition, 50, 11-18.

Ma, J.F. (2009) Silicon uptake and translocation in plants. The Proceedings of the

International Plant Nutrition Colloquium XVI. Department of Plant Sciences, UC

Davis, UC Davis.

Ma, J.F., Tamai, K., Yamaji, N., Mitani, N., Konishi, S., Katsuhara, M., Ishiguro, M.,

Murata, Y. & Yano, M. (2006) A silicon transporter in rice. Nature, 440, 688-691.

Ma, J.F. & Yamaji, N. (2006) Silicon uptake and accumulation in higher plants. Trends in

Plant Science, 11, 392-397.

Ma, J.F. & Yamaji, N. (2015) A cooperative system of silicon transport in plants. Trends in

Plant Science, 20, 435-442.

31

726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

747

748

Massey, F.P., Ennos, A.R. & Hartley, S.E. (2006) Silica in grasses as a defence against insect

herbivores: contrasting effects on folivores and a phloem feeder. Journal of Animal

Ecology, 75, 595-603.

Massey, F.P., Ennos, A.R. & Hartley, S.E. (2007a) Grasses and the resource availability

hypothesis: the importance of silica-based defences. Journal of Ecology, 95, 414-424.

Massey, F.P., Ennos, A.R. & Hartley, S.E. (2007b) Herbivore specific induction of silica-

based plant defences. Oecologia, 152, 677-683.

Massey, F.P. & Hartley, S.E. (2006) Experimental demonstration of the antiherbivore effects

of silica in grasses: impacts on foliage digestibility and vole growth rates.

Proceedings of the Royal Society B-Biological Sciences, 273, 2299-2304.

Massey, F.P. & Hartley, S.E. (2009) Physical defences wear you down: progressive and

irreversible impacts of silica on insect herbivores. Journal of Animal Ecology, 78,

281-291.

Massey, F.P., Massey, K., Ennos, A.R. & Hartley, S.E. (2009) Impacts of silica-based

defences in grasses on the feeding preferences of sheep. Basic and Applied Ecology,

10, 622-630.

Massey, F.P., Smith, M.J., Lambin, X. & Hartley, S.E. (2008) Are silica defences in grasses

driving vole population cycles? Biology letters, 4, 419-422.

McArt, S.H., Spalinger, D.E., Collins, W.B., Schoen, E.R., Stevenson, T. & Bucho, M.

(2009) Summer dietary nitrogen availability as a potential bottom-up constraint on

moose in south-central Alaska. Ecology, 90, 1400-1411.

McArthur, C. (2014) Do we ditch digestive physiology in explaining the classic relationship

between herbivore body size diet and diet quality? Functional Ecology, 28, 1059-

1060.

32

749

750

751

752

753

754

755

756

757

758

759

760

761

762

763

764

765

766

767

768

769

770

771

772

McColloch, J.W. & Salmon, S.C. (1923) The resistance of wheat to the Hessian Fly - a

progress report. Journal of Economic Entomology, 16, 293-298.

McNaughton, S.J. & Tarrants, J.L. (1983) Grass leaf silicification - natural selection for an

inducible defense against herbivores. Proceedings of the National Academy of

Sciences of the United States of America-Biological Sciences, 80, 790-791.

McNaughton, S.J., Tarrants, J.L., McNaughton, M.M. & Davis, R.H. (1985) Silica as a

defense against herbivory and a growth promotor in African grasses. Ecology, 66,

528-535.

Moraes, J.C., Goussain, M.M., Basagli, M.A.B., Carvalho, G.A., Ecole, C.C. & Sampaio,

M.V. (2004) Silicon influence on the tritrophic interaction: Wheat plants, the

greenbug Schizaphis graminum (Rondani) (Hemiptera : Aphididae), and its natural

enemies, Chrysoperla externa (Hagen) (Neuroptera : Chrysopidae) and Aphidius

colemani viereck (Hymenoptera : Aphidiidae). Neotropical Entomology, 33, 619-624.

Piperno, D.R. (2006) Pytoliths: A comprehensive Guide for Archaeologists and

Palaeoecologists. AltaMira Press, Oxford.

Ponnaiya, B. (1951) Studies on the genus Sorghum. II The cause of resistance in sorghum to

the insect pest Atherigona indica M. Madras University Journal, 21, 203-217.

Quigley, K.M. & Anderson, T.M. (2014) Leaf silica concentration in Serengeti grasses

increases with watering but not clipping: insights from a common garden study and

literature review. Frontiers in Plant Science, 5, 568.

Rabenold, D. & Pearson, O.M. (2011) Abrasive, silica phytoliths and the evolution of thick

molar enamel in primates, with implications for the diet of Paranthropus boisei. Plos

One, 6, e28379.

Raven, J.A. (1983) The transport and function of silicon in plants. Biological Reviews of the

Cambridge Philosophical Society, 58, 179-207.

33

773

774

775

776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

793

794

795

796

797

Redmond, C.T. & Potter, D.A. (2006) Silicon fertilization does not enhance creeping

bentgrass resistance to cutworms and white grubs. Applied Turfgrass Science

[Online], 6, 1-7.

Reidinger, S., Ramsey, M.H. & Hartley, S.E. (2012) Rapid and accurate analyses of silicon

and phosphorus in plants using a portable X-ray fluorescence spectrometer. New

Phytologist, 195, 699-706.

Reymond, P., Weber, H., Damond, M. & Farmer, E.E. (2000) Differential gene expression in

response to mechanical wounding and insect feeding in Arabidopsis. Plant Cell, 12,

707-719.

Reynolds, J.J.H., Lambin, X., Massey, F.P., Reidinger, S., Sherratt, J.A., Smith, M.J., White,

A. & Hartley, S.E. (2012) Delayed induced silica defences in grasses and their

potential for destabilising herbivore population dynamics. Oecologia, 170, 445-456.

Reynolds, O.L., Keeping, M.G. & Meyer, J.H. (2009) Silicon-augmented resistance of plants

to herbivorous insects: a review. Annals of Applied Biology, 155, 171-186.

Rivals, F., Takatsuki, S., Maria Albert, R. & Macia, L. (2014) Bamboo feeding and tooth

wear of three sika deer (Cervus nippon) populations from northern Japan. Journal of

Mammalogy, 95, 1043-1053.

Sangster, A.G., Hodson, M.J. & Tubb, H.J. (2001) Silicon deposition in higher plants. Silicon

in Agriculture (eds L.E. Datnoff, G.H. Snyder & G.H. Korndorfer), pp. 85-113.

Elsevier Science, Amsterdam.

Sanson, G.D., Kerr, S.A. & Gross, K.A. (2007) Do silica phytoliths really wear mammalian

teeth? Journal of Archaeological Science, 34, 526-531.

Sasamoto, K. (1953) Studies on the relation between insect pests and silica content in rice

plant (II). On the injury of the second generation larvae of rice stem borer. Oyo

Kontyu, 9, 108-110.

34

798

799

800

801

802

803

804

805

806

807

808

809

810

811

812

813

814

815

816

817

818

819

820

821

822

Seastedt, T.R., Ramundo, R.A. & Hayes, D.C. (1989) Silica, nitrogen and phosphorus

dynamics of tallgrass prairie. 11th North American Prairie Conference, pp. 205-210.

University of Nebraska Press.

Shewmaker, G.E., Mayland, H.F., Rosenau, R.C. & Asay, K.H. (1989) Silicon in C3 grasses -

effects on forage quality and sheep preference. Journal of Range Management, 42,

122-127.

Smith, M.J., White, A., Lambin, X., Sherratt, J.A. & Begon, M. (2006) Delayed density-

dependent season length alone can lead to rodent population cycles. American

Naturalist, 167, 695-704.

Soininen, E.M., Brathen, K.A., Jusdado, J.G.H., Reidinger, S. & Hartley, S.E. (2013) More

than herbivory: levels of silica-based defences in grasses vary with plant species,

genotype and location. Oikos, 122, 30-41.

Stam, J.M., Kroes, A., Li, Y., Gols, R., van Loon, J.J.A., Poelman, E.H. & Dicke, M. (2014)

Plant interactions with multiple insect herbivores: From community to genes. Annual

Review of Plant Biology, 65, 689-713.

Steuer, P., Suedekum, K.-H., Tuetken, T., Mueller, D.W.H., Kaandorp, J., Bucher, M.,

Clauss, M. & Hummel, J. (2014) Does body mass convey a digestive advantage for

large herbivores? Functional Ecology, 28, 1127-1134.

Strömberg, C., Di Stilio, V. & Song, Z. (2016) Functions of phytoliths in vascular plants: An

evolutionary perspective. Functional Ecology, In press.

Tanentzap, A.J., Vicari, M. & Bazely, D.R. (2014) Ungulate saliva inhibits a grass-endophyte

mutualism. Biology letters, 10.

Tian, D., Peiffer, M., Shoemaker, E., Tooker, J., Haubruge, E., Francis, F., Luthe, D.S. &

Felton, G.W. (2012) Salivary glucose oxidase from caterpillars mediates the induction

of rapid and delayed-induced defenses in the tomato plant. Plos One, 7, e36168.

35

823

824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

846

847

Valkama, E., Koricheva, J., Ossipov, V., Ossipova, S., Haukioja, E. & Pihlaja, K. (2005)

Delayed induced responses of birch glandular trichomes and leaf surface lipophilic

compounds to mechanical defoliation and simulated winter browsing. Oecologia, 146,

385-393.

Vicari, M. & Bazely, D.R. (1993) Do grasses fight back? The case for antiherbivore

defences. Trends in Ecology & Evolution, 8, 137-141.

Walters, D. (2010) Plant Defense: Warding off Attack by Pathogens, Herbivores and

Parasitic Plants. Wiley-Blackwell.

Watling, K.M., Parr, J.F., Rintoul, L., Brown, C.L. & Sullivan, L.A. (2011) Raman, infrared

and XPS study of bamboo phytoliths after chemical digestion. Spectrochimica Acta

Part A-Molecular and Biomolecular Spectroscopy, 80, 106-111.

Wieczorek, M., Szafranska, P.A., Labecka, A.M., Lazaro, J. & Konarzewski, M. (2015a)

Effect of the abrasive properties of sedges on the intestinal absorptive surface and

resting metabolic rate of root voles. Journal of Experimental Biology, 218, 309-315.

Wieczorek, M., Zub, K., Szafranska, P.A., Ksiazek, A. & Konarzewski, M. (2015b) Plant-

herbivore interactions: silicon concentration in tussock sedges and population

dynamics of root voles. Functional Ecology, 29, 187-194.

Yamaji, N., Sakurai, G., Mitani-Ueno, N. & Ma, J.F. (2015) Orchestration of three

transporters and distinct vascular structures in node for intervascular transfer of

silicon in rice. Proceedings of the National Academy of Sciences of the United States

of America, 112, 11401-11406.

Ye, M., Song, Y., Long, J., Wang, R., Baerson, S.R., Pan, Z., Zhu-Salzman, K., Xie, J., Cai,

K., Luo, S. & Zeng, R. (2013) Priming of jasmonate-mediated antiherbivore defense

responses in rice by silicon. Proceedings of the National Academy of Sciences of the

United States of America, 110, E3631-E3639.

36

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

865

866

867

868

869

870

871

872

Figure legends

Figure 1

Induction of silica defences in Deschampsia caespitosa exposed to high (~20% of leaves

damaged) and low (~5% of leaves damaged) levels of grazing by field voles (Microtus

agrestis). Solid lines denote high grazing intensity and broken lines denote low grazing

intensity. (a) Comparison of silica induction in D. caespitosa grown in glasshouse and grazed

in the laboratory (reproduced from Reynolds et al. 2012) and under field conditions in open

grazing enclosures in northern England from May-November 2009 with a grazing intensity of

300 voles/ha and 50 voles/ha (J. DeGabriel, S. Hartley, F. Massey, S. Reidinger and X.

Lambin, unpublished data). (b) Seasonal variation in silica concentrations in D. caespitosa in

field grazing enclosures under high (300 voles/ha) and low (50 voles/ha) grazing treatments

from March 2009-April 2010. Error bars represent standard error.

Figure 2

A summary of research needs for silicon-mediated ecological interactions between

plants and herbivores. Green boxes summarise established knowledge, whilst the pink

boxes suggest key knowledge gaps and potential research questions for future work, as

depicted by the graphics in circles.

37

873

874

875

876

877

878

879

880

881

882

883

884

885

886

887

888

889

890

891

0 1 2 3 4 5 60

1

2

3

4

5

6

7

8

Month

Mea

n sil

ica

conc

entr

ation

(%DM

)

Spring Summer Autumn Winter Spring0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Season

Mea

n sil

ica

conc

entr

ation

(% D

M)

38

892

893

894

39

895

896